microRNAs (miRNAs) comprise a family of regulatory RNAs that repress the expression of target messenger RNAs. The early embryonic lethality caused by loss of miRNAs highlights their requirement for normal mammalian development, and altered miRNA expression is observed in cancer. Mature ~22 nucleotide miRNAs are produced from long primary transcripts through sequential cleavages by the Microprocessor and Dicer complexes. One strand of the miRNA duplex is incorporated into the RNA induced silencing complex (RISC) and directs mRNA cleavage or translational repression. Over 500 miRNAs have been identified, many of which are expressed in a developmentally regulated manner. Under most conditions, control of their expression occurs at the transcriptional level. However, it is emerging that miRNA processing can also be regulated. Certain miRNA precursors are highly expressed in embryonic stem (ES) cells, and primary tumors even though the corresponding mature species are undetectable. This suggests a block in miRNA-processing, the mechanism of which has remained elusive. Our goal is to understand the mechanism by which miRNA processing is regulated in ES cells. We recently identified the developmentally regulated RNA-binding protein Lin28 as a selective inhibitor of miRNA processing. Our preliminary data indicate that Lin28 is a component of a large multi-subunit complex in ES cells. To gain insight into the mechanism of Lin28 function we plan to isolate Lin28 (and Lin28B) protein complexes from ES cells and identify and functionally characterize complex components. We will employ a variety of approaches to identify the RNA component of Lin28-containing ribonucleoprotein complexes. Once we have confirmed the biochemical association of particular polypeptides and RNAs with Lin28 we will examine their subcellular localization in ES cells using both immunolocalization and in-situ hybridization analyses. Our current data indicate that Lin28 selectively inhibits processing of a particular subset of miRNAs, however the determinants and mechanism of this regulatory pathway remain unknown. We will perform detailed biochemical analyses including in vitro reconstitution experiments and mutagenesis of critical Lin28 residues to functionally define important protein domains. We seek to understand the RNA structural and sequence determinants of processing inhibition and will generate artificial miRNA precursors and measure processing inhibition by Lin28. Additionally, we plan to functionally characterize the role of Lin28 complex components in the regulation of miRNA processing in ES cells. Accomplishing the goals of this proposal will provide a better understanding of how stem cells are regulated at the molecular level and could aid in the development of novel therapeutics. PUBLIC HEALTH REVELANCE: Stem cells hold great promise for the development of new approaches to combat disease. However, the molecular basis for stem cell self-renewal and differentiation remains incompletely understood. The proposed work will provide novel insight into embryonic stem cell gene regulation, and may lead to new therapies to manipulate microRNAs. These studies are relevant to the treatment of cancer, diabetes, developmental disorders and numerous degenerative diseases.